The immunobiology of HIV-1 infection in the female genital tract involves three major events: (a) Entry through the mucosal epithelium; (b) Infection and subsequent replication in subepithelial mononuclear cells; and (c) Delivery to lymph nodes to initiate systemic infection. To dissect these events, studies of macaque and human genital tissues have yielded variable results, likely because multiple cells may be involved in the events. Moreover, microbicidal agents that block SIV infection in macaques fail to block, or even enhance, infection in women, underscoring the urgent need for an effective mucosal vaccine. Importantly, because women may also acquire HIV-1 through rectal exposure, an effective vaccine for all at-risk women needs to block HIV-1 entry and infection in both genital and rectal mucosa. Using a mucosal explant system and purified mucosal cells, this proposal will elucidate the immunobiology of HIV-1 entry and infection and characterize the effect of anti-HIV-1 antibodies and progesterone-based hormonal agents on these events in genital and rectal mucosa. The proposal is driven by four Hypotheses: (1) HIV-1 crosses the monostratified endocervical and rectal epithelium by epithelial cell transcytosis but crosses pleuristratified ectocervical and vaginal epithelium via DCs; (2) Female genital mucosa selects the R5, genotypically restricted viruses that characterize acute HIV-1 infection; (3) In female genital mucosa, macrophages, lymphocytes and DCs are permissive to HIV-1 infection, but in rectal mucosa only lymphocytes and DCs are permissive; and (4) HIV- 1 entry and infection in genital and rectal mucosae are inhibited by IgG (and possibly IgA) anti-HIV-1 antibodies, and receptor analogs and ligands. These hypotheses will be tested with four Specific Aims: (1) Determine the cell(s), attachment molecule(s) and receptor(s) that cell-free and cell-associated R5 and X4 HIV-1 utilizes to enter the endocervical, ectocervical, vaginal and rectal epithelium. (2) Determine whether genital mucosa selects the R5 viruses that characterize acute HIV-1 infection. (3) Determine whether HIV-1 in female genital mucosa infects lymphocytes, macrophages and DCs but in rectal mucosa infects only lymphocytes and DCs. (4) Determine whether anti-HIV-1 (gp41 GalCer-binding domain, gp41, gp120) antibodies and CCR5 anti-virals block cell-free and cell-associated R5 and X4 HIV-1 entry and target cell infection in female genital and rectal mucosa. RELEVANCE (See instructions): This proposal will use primary mucosal tissues and purified mucosal cells to define the key events in HIV-1 entry and infection in human female genital and rectal tissue in the presence and absence of progesterone- based hormonal agents. The ability of antibodies and anti-receptor agents to block these events will be characterized, thereby providing critical information for the development of a mucosal vaccine to prevent genital and rectal HIV-1 infection in women.